Giant Axonal Neuropathy (GAN) is an autosomal recessive neurodegenerative disease. In these patients, degeneration of motor neurons (MNs) leads to progressive paralysis and death by the third decade of life. GAN patients have mutations in the gene coding for gigaxonin, thought to be important for the proteasome- dependent degradation of several cytoskeletal proteins. Biopsies of GAN patients are characterized by the presence of accumulations of neurofilament proteins (NFs) in neurons and intermediate filaments (IFs) in a variety of other cell types, including astrocytes and Schwann cells. GAN knock-out mice do not have an overt neurological phenotype and do not develop giant axons as seen in the human disease; moreover, human MNs are not accessible to biopsy and subsequent culture. Therefore, I propose to generate a human model of this disease using induced pluripotent stem cells (iPSCs). The first aim of my study will be to generate new tools to study GAN, including induced pluripotent stem cell- derived motor neurons (iPS-MNs) from GAN patients. To date I have generated at least one iPS line from each of four GAN patients. I also propose to generate reporter lines to identify MNs in live cultures and rescue lines to study the effects of a loss of gigaxonin on an isogenetic background. A rescue lentivirus will be utilized to determine if replacement of gigaxonin can reverse any GAN-related phenotypes, as a first proof of concept for gene therapy. GAN and control iPSCs will be differentiated into MNs and characterized for MN markers and activity. In the second aim, I will use these GAN iPS-MNs to examine biochemical, cell biological, and functional alterations in developing GAN iPS-MNs and their gigaxonin-dependence. I will investigate the mechanism(s) responsible for the elevated NF levels I observe, and any additional GAN-related phenotypes. As in GAN patients, I find that levels of IFs such as peripherin and NF-L are significantly elevated in GAN iPS-MN cultures. I have found two populations of peripherin expressing cells in GAN iPS-MN cultures; those that express peripherin similar to control MNs and those that express elevated levels. In addition, GAN iPS-MNs have a 5-fold incidence in the occurrence of peripherin positive perinuclear aggregates. I will continue this analysis and will assess for other function consequences from loss of gigaxonin, including morphological and survival changes, and the extent to which these phenotypes can be reversed by replacement of gigaxonin. Lastly, I will investigate the mechanism(s) behind the elevated peripherin levels I observe by immunoprecipitation of FLAG-tagged gigaxonin from human motor neurons to determine its key interacting partners. This project represents the first GAN model based on the study of human motor neurons and should inform our understanding the pathogenesis of GAN and other neurodegenerative diseases of the cytoskeleton. PUBLIC HEALTH RELEVANCE: In the autosomal recessive disease Giant Axonal Neuropathy (GAN), degeneration of motor neurons (MNs) leads to progressive paralysis and death by the third decade of life. This disease is poorly understood and currently incurable. Because the mouse model does not phenocopy the human disease and human MNs are inaccessible to biopsy, I propose to generate a human model of this disease using induced pluripotent stem cells (iPSCs). This model will provide a platform to test for viral rescue on human MNs, which may provide the first proof of principle for the treatment of GAN using gene therapy.